HHS Public Access Author manuscript Author Manuscript
Nat Methods. Author manuscript; available in PMC 2017 August 13. Published in final edited form as: Nat Methods. 2017 February 13; 14(4): 399–402. doi:10.1038/nmeth.4178.
Atomic resolution structures from fragmented protein crystals by the cryoEM method MicroED M. Jason de la Cruz1, Johan Hattne1, Dan Shi1, Paul Seidler2, Jose Rodriguez2, Francis E. Reyes1, Michael R. Sawaya2, Duilio Cascio2, Simon C. Weiss3, Sun Kyung Kim3, Cynthia S. Hinck3, Andrew P. Hinck3, Guillermo Calero3, David Eisenberg2, and Tamir Gonen1,* 1Howard
Hughes Medical Institute, Janelia Research Campus, Ashburn, Virginia 20147, USA
Author Manuscript
2Howard
Hughes Medical Institute, UCLA-DOE Institute, Departments of Biological Chemistry, Chemistry & Biochemistry, and Molecular Biology Institute, Box 951570, UCLA, Los Angeles, California 90024, USA
3University
of Pittsburgh, Department of Structural Biology, 3501 Fifth Avenue, Pittsburgh, Pennsylvania 15260, USA
Abstract
Author Manuscript
Crystallographic analysis of macromolecules depends on large, well-ordered crystals, which often require significant effort to obtain. Even sizable crystals sometimes suffer from pathologies that render them inappropriate for high-resolution structure determination. Here we show that fragmentation of large, imperfect crystals can provide a simple path for high-resolution structure determination by serial femtosecond crystallography or the cryoEM method MicroED.
Introduction Large and perfect crystals are desirable for structure determination because they yield a strong signal over background. In reality, crystals of biological material are not perfect. In the mosaic model, a real, imperfect crystal is composed of several small but well-ordered blocks1. These mosaic blocks have a finite size, are misaligned with respect to each other,
Author Manuscript
Users may view, print, copy, and download text and data-mine the content in such documents, for the purposes of academic research, subject always to the full Conditions of use:http://www.nature.com/authors/editorial_policies/license.html#terms * To whom correspondence should be addressed T.G. ([email protected]). Author contributions DS and TG designed experiment; MJdlC and FER prepared lysozyme, xylanase, thaumatin, trypsin, proteinase K, and thermolysin samples; JR and DE prepared tau peptide samples; SCW, SKK, CSH, APH, and CG prepared TGF-βm:TβRII samples; MJdlC, DS, JR, and SCW collected data; MJdlC, JH, PS, MRS, DC, SCW, and GC, analyzed data, refined and determined models; JH, JR, and MRS prepared figures; MJdlC, JH, and TG wrote the manuscript with contributions from all authors. Competing financial interests statement The authors declare no competing financial interests. Accession codes Data availability statement Atomic coordinates and structure factors were deposited to the Protein Data Bank (PDB; 5k7n, 5k7o, 5ty4, 5k7p, 5k7q, 5k7r, 5k7s, and 5k7t) and the Electron Microscopy Data Bank (EMD; EMD-8216, EMD-8217, EMD-8472, EMD-8218, EMD-8219, EMD-8220, EMD-8221, and EMD-8222), and the raw data were uploaded to the Structural Biology Data Grid38 (10.15785/SBGRID/284, 10.15785/SBGRID/285, 10.15785/SBGRID/368, 10.15785/SBGRID/286, 10.15785/SBGRID/287, 10.15785/SBGRID/288, 10.15785/ SBGRID/289, 10.15785/SBGRID/290).
de la Cruz et al.
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and may be composed of unit cells with different dimensions2. Depending on the nature and degree of disorder between the mosaic blocks, an imperfect crystal may exhibit a plethora of pathologies, which may hamper subsequent data reduction, limit the resolution of the final model, and can prevent structure determination altogether (Supplementary figures 1–9). Many such defects primarily affect larger crystals, and small crystals may yield superior data quality where diffraction is not limited by the number of diffracting unit cells in the crystal3.
Author Manuscript
For these reasons, methods such as serial femtosecond crystallography (SFX)4 at an X-ray free-electron laser (XFEL) and the electron cryomicroscopy (cryoEM) method microelectron diffraction (MicroED)5 are actively being developed. These methods can yield structures to resolutions better than 1 Å from crystals that are significantly smaller than those which are required for standard crystallography: ~10,000× smaller in volume for XFEL and ~1,000,000× smaller for MicroED. In fact, in both these techniques large crystals can prove problematic. This is because large crystals can clog up the nozzle of certain SFX sample delivery systems6,7, while in MicroED the large electron scattering cross-section implies that absorption extinguishes diffraction when the sample is too thick8.
Author Manuscript Author Manuscript
Here we show that sonication, vigorous pipetting, or vortexing can be used to break large imperfect crystals into small, single-crystal fragments that are suitable for data collection and atomic structure determination (Figure 2). Delicate samples may benefit from gentler fragmentation by vortexing, while harsher methods such as pipetting and ultimately sonication are required to break more robust crystals. In many cases, the untreated crystals were mosaic, yielded diffraction patterns with multiple lattices, or were otherwise not suitable for standard crystallographic experiments (Figure 2, Supplementary figures 1–9). In the case of the amyloid-forming peptide of tau (VQIVYK), what appeared to be large crystals were in fact crystal bundles that produced low resolution powder-like diffraction. These problems are traditionally overcome by modifying the crystallization conditions to optimize crystal growth and quality a process that can be tedious and labor intensive, particularly when crystal pathologies do not become apparent until data processing. Breaking of large crystals by physical means produced fragments that appeared crystallographically homogeneous and yielded diffraction data at atomic resolution, void of the above artifacts. This was true even without further optimization of the growth conditions for crystals formed by various macromolecules of molecular weights between 0.7 kDa and 34.6 kDa, and solvent contents ranging from 30% to 60% with a range of unit cell dimensions and space groups (Table 1). MicroED has already allowed rapid structure solution from several peptide fragments that resisted solution using microfocus synchrotron sources in spite of many months of crystal optimization (Supplementary figures 2–4), and the approach could be used for samples where large crystals exist but standard crystallographic methods fail, owing to crystal imperfections. Fragmentation was tested on eight proteins: lysozyme, TGF-βm:TβRII, xylanase, thaumatin, trypsin, proteinase K, thermolysin, and a segment of the protein tau (Figure 2, left column). These proteins readily form crystals that are large—too big for MicroED and SFX experiments using liquid injectors—but without crystal growth optimization sometimes exhibit pathologies. These large crystals were broken apart by mechanical means prior to preparation on cryoEM grids. Micro- or nanometer-sized crystal fragments appeared evenly
Nat Methods. Author manuscript; available in PMC 2017 August 13.
de la Cruz et al.
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distributed on the grid, and even when grids were densely populated single-crystal data sets could be collected by using the selective area aperture. MicroED is inherently well suited to study crystal fragments. Electrons interact strongly with matter so that large crystals are not required to detect high-resolution Bragg reflections8. MicroED has been used to determine the structures of the enzymes lysozyme5,9, proteinase K10, catalase11,12, and Ca2+-ATPase12. It was also recently used to solve the 1.4 Å resolution structure of the toxic NAC-core of α-synuclein, where diffraction data were collected from crystals smaller than the wavelength of visible light13. We previously demonstrated that crystals thinner than ~400 nm are suitable for MicroED and routinely yield atomic resolution information. The highest resolution structures reported so far by using MicroED are from four prion protein fragments determined at 1 Å resolution solved by direct phasing methods14.
Author Manuscript Author Manuscript
For each sample, data were collected from 2–10 crystal fragments, and data sets merged for completeness and multiplicity (Table 1). Although we have previously published two structures where a single nanocrystal was sufficient for structure determination9,11 multicrystal merging is generally preferred. This is partly because the grid on which the crystals are mounted limits the accessible rotation range during data collection, and partly because small, weakly scattering samples require a more intense beam to yield statistically accurate measurements of the Bragg reflections, and may be irreversibly damaged before a complete data set can be obtained. Recording narrow wedges with fewer exposures for each crystal involves a tradeoff between limiting the damage to the sample and maintaining a sufficient dose rate for the signal from high-resolution reflections to be accurately integrated. Because the resolutions obtained by MicroED are comparable or better to what was obtained by Xray diffraction before sonication, it appears that fragmentation only broke apart the large crystals into small crystal domains but did not otherwise damage the lattice order.
Author Manuscript
The macromolecular structures in this study were phased using molecular replacement (MR) or by direct methods and refined using electron scattering factors15. In all cases, the refined model fits the calculated density well (Figure 2, middle column) with an overall real-space map correlation coefficient ranging from 0.72 to 0.91 (RSCC; Table 1). The resulting simulated-annealing (SA) composite-omit maps (Figure 2, right column) match their respective models very well, indicating that the data are indeed high quality and unbiased. Further, the SA omit maps for lysozyme reveal depressions or holes in the aromatic rings of amino acid side chains, while for proteinase K toroidal densities were observed even for proline residues at the 1.6 Å resolution cutoff. A well-coordinated calcium ion is clearly visible in the omit map for trypsin, as is one of the iodides in the xylanase structure. Individual atoms are visible in the density for the tau peptide, and several residues show positive 2mFo-DFc peaks for hydrogen atoms; for one residue, modeled hydrogens allowed us to unambiguously place the correct sidechain rotamer. All structures were determined between 1.1–2.9 Å resolution. We note that the R-factors from model refinement are generally higher in MicroED than typical values obtained in Xray crystallography at similar resolutions. Others and we have already made such observations in previously published studies5,9,12. We believe that the main reason is lack of
Nat Methods. Author manuscript; available in PMC 2017 August 13.
de la Cruz et al.
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adequate electron scattering factors in the crystallographic refinement software. X-rays are scattered by the electron cloud of an atom while electrons are scattered by the atomic Coulomb potential, which arises from the nucleus as well as its surrounding electrons. Current electron scattering factor tables do not adequately account for this difference and this may contribute to the residual between observed and calculated structure factor amplitudes leading to higher than normal crystallographic and free R-factors. We note that the gap between Rwork and Rfree is small indicating no concerns of overfitting (Supplementary figure 10).
Discussion
Author Manuscript
Breaking large crystals into well-formed crystal fragments has a long history in crystallography; indeed X-ray diffraction was discovered from broken up pieces of a copper sulfate crystal16. Sonication17 and vortexing18 have previously been used to prepare crystals of suitable size for XFEL measurements. By breaking up one imperfect large crystal into thousands or even millions of smaller crystallites and recombining a well-diffracting subset during data processing, we have used MicroED to determine the structure of eight different proteins to high resolution. Fragmentation does not increase the effort required for sample screening, and future work on automation is expected to reduce the time for data collection irrespective of how the sample was prepared. Importantly, as demonstrated by the breadth of size and packing of macromolecules whose crystals we investigated, this is a broadly applicable approach where large crystals are available. Fragmentation widens the scope of diffraction methods such as MicroED and SFX to include samples that do not exclusively form tiny crystals but instead form large, imperfect crystals.
Author Manuscript
Online methods Large crystals (>500 µm along the longest edge) were grown via hanging-drop vapor diffusion at room temperature using previously established protocols. All enzymes were purchased from Sigma-Aldrich (St. Louis, MO) unless otherwise noted.
Author Manuscript
•
Tau peptide (VQIVYK) was dissolved in distilled water and crystals prepared by mixing tau peptide with arachidonic acid and meclocycline sulfosalicylate. Crystals formed with a 1:2 drop peptide to precipitant ratio from 65–70% ethylene glycol in Tris pH 8.5.
•
Lysozyme (G. gallus) crystals were prepared by equilibrating equal volumes of 50 mg/ml lysozyme in water and 1.7 M sodium chloride, 50 mM sodium acetate pH 4.7.
•
TGF-βm:TβRII was expressed and purified as described before20,21. Crystals were grown from 0.5 µl of protein (20 mg/ml), 0.25 µl mother liquor, and 0.2 µl seed stock in 100 mM HEPES/NaOH pH 7.5 and 45% MPD.
•
Xylanase from T. reesei (Hampton Research, Aliso Viejo, CA) was dialyzed against 10 mM bicine pH 9.0, 1 mM magnesium sulfate, 1 mM DTT and combined with a precipitant solution containing 0.3 M sodium iodide, 1.2–1.3 M ammonium sulfate, and 100 mM bicine pH 9.022.
Nat Methods. Author manuscript; available in PMC 2017 August 13.
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•
Thaumatin (T. daniellii) crystals were grown from 2 µL of protein (25 mg/ml in water) and Hampton Research Index Reagent 26 (1.1 M ammonium tartate pH 7.0).
•
Trypsin (B. taurus) was dissolved (60 mg/ml) in 10 mg/ml benzamidine, 3 mM calcium chloride and equilibrated against 4% (w/v) PEG4000, 0.2 M lithium sulfate, 0.1 M MES pH 6.5 and 15% ethylene glycol.
•
Proteinase K (E. album) crystals were grown by combining 2 µL of protein solution (50 mg/ml) with 2 µL of precipitant solution (1.0–1.3 M ammonium sulfate, 0.1 M Tris pH 8.0)10.
•
Thermolysin from B. thermoproteolyticus (Hampton Research, Aliso Viejo, CA) crystals were prepared by equilibrating a 160 mg/ml solution of thermolysin (45% dimethyl sulfoxide, 50 mM Tris pH 7.5 and 2.5 M cesium chloride) over 0.5 ml of water23.
Drops containing the crystals were placed in separate microfuge tubes and suspended in crystal mother liquor. A sonicating water bath with electronic control (Elmasonic P30H, Singen, Germany) was set at its lowest power (30% at 37 kHz) for gentle agitation of the crystals in the tube. With the tube sealed, its tip was briefly submerged in the activated water bath for 0.5 s. Alternatively; crystals were fragmented by vigorously pipetting a crystal suspension in mother liquor (trypsin and thaumatin) or vortexed with 0.5 mm disruption glass beads in a 1.5 ml reaction tube for 2 s (TGF-βm:TβRII). A detailed protocol for crystal fragmentation was published online24.
Author Manuscript Author Manuscript
The solution containing fragmented crystals was then applied to transmission electron microscope (TEM) grids with carbon film support, and plunge-frozen in liquid ethane. Frozen grids were mounted in a Gatan Model 626 cryo-specimen holder and examined using an FEI Tecnai F20 field-emission TEM operated at an accelerating voltage of 200 kV, which corresponds to a de Broglie wavelength of 0.025 Å, and screened for crystals in overfocused diffraction mode. Where a single still shot revealed strong diffraction, data were collected as continuous rotation tilt series9. Individual frames were recorded on a TVIPS TemCam-F416 as 4 s exposures while the stage was rotating at 0.09°/s, except for tau peptide, which was rotated at 0.29°/s during 2 s exposures. For the macromolecular samples, the absolute tilt angle was generally
Nat Methods. Author manuscript; available in PMC 2017 August 13. Published in final edited form as: Nat Methods. 2017 February 13; 14(4): 399–402. doi:10.1038/nmeth.4178.
Atomic resolution structures from fragmented protein crystals by the cryoEM method MicroED M. Jason de la Cruz1, Johan Hattne1, Dan Shi1, Paul Seidler2, Jose Rodriguez2, Francis E. Reyes1, Michael R. Sawaya2, Duilio Cascio2, Simon C. Weiss3, Sun Kyung Kim3, Cynthia S. Hinck3, Andrew P. Hinck3, Guillermo Calero3, David Eisenberg2, and Tamir Gonen1,* 1Howard
Hughes Medical Institute, Janelia Research Campus, Ashburn, Virginia 20147, USA
Author Manuscript
2Howard
Hughes Medical Institute, UCLA-DOE Institute, Departments of Biological Chemistry, Chemistry & Biochemistry, and Molecular Biology Institute, Box 951570, UCLA, Los Angeles, California 90024, USA
3University
of Pittsburgh, Department of Structural Biology, 3501 Fifth Avenue, Pittsburgh, Pennsylvania 15260, USA
Abstract
Author Manuscript
Crystallographic analysis of macromolecules depends on large, well-ordered crystals, which often require significant effort to obtain. Even sizable crystals sometimes suffer from pathologies that render them inappropriate for high-resolution structure determination. Here we show that fragmentation of large, imperfect crystals can provide a simple path for high-resolution structure determination by serial femtosecond crystallography or the cryoEM method MicroED.
Introduction Large and perfect crystals are desirable for structure determination because they yield a strong signal over background. In reality, crystals of biological material are not perfect. In the mosaic model, a real, imperfect crystal is composed of several small but well-ordered blocks1. These mosaic blocks have a finite size, are misaligned with respect to each other,
Author Manuscript
Users may view, print, copy, and download text and data-mine the content in such documents, for the purposes of academic research, subject always to the full Conditions of use:http://www.nature.com/authors/editorial_policies/license.html#terms * To whom correspondence should be addressed T.G. ([email protected]). Author contributions DS and TG designed experiment; MJdlC and FER prepared lysozyme, xylanase, thaumatin, trypsin, proteinase K, and thermolysin samples; JR and DE prepared tau peptide samples; SCW, SKK, CSH, APH, and CG prepared TGF-βm:TβRII samples; MJdlC, DS, JR, and SCW collected data; MJdlC, JH, PS, MRS, DC, SCW, and GC, analyzed data, refined and determined models; JH, JR, and MRS prepared figures; MJdlC, JH, and TG wrote the manuscript with contributions from all authors. Competing financial interests statement The authors declare no competing financial interests. Accession codes Data availability statement Atomic coordinates and structure factors were deposited to the Protein Data Bank (PDB; 5k7n, 5k7o, 5ty4, 5k7p, 5k7q, 5k7r, 5k7s, and 5k7t) and the Electron Microscopy Data Bank (EMD; EMD-8216, EMD-8217, EMD-8472, EMD-8218, EMD-8219, EMD-8220, EMD-8221, and EMD-8222), and the raw data were uploaded to the Structural Biology Data Grid38 (10.15785/SBGRID/284, 10.15785/SBGRID/285, 10.15785/SBGRID/368, 10.15785/SBGRID/286, 10.15785/SBGRID/287, 10.15785/SBGRID/288, 10.15785/ SBGRID/289, 10.15785/SBGRID/290).
de la Cruz et al.
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and may be composed of unit cells with different dimensions2. Depending on the nature and degree of disorder between the mosaic blocks, an imperfect crystal may exhibit a plethora of pathologies, which may hamper subsequent data reduction, limit the resolution of the final model, and can prevent structure determination altogether (Supplementary figures 1–9). Many such defects primarily affect larger crystals, and small crystals may yield superior data quality where diffraction is not limited by the number of diffracting unit cells in the crystal3.
Author Manuscript
For these reasons, methods such as serial femtosecond crystallography (SFX)4 at an X-ray free-electron laser (XFEL) and the electron cryomicroscopy (cryoEM) method microelectron diffraction (MicroED)5 are actively being developed. These methods can yield structures to resolutions better than 1 Å from crystals that are significantly smaller than those which are required for standard crystallography: ~10,000× smaller in volume for XFEL and ~1,000,000× smaller for MicroED. In fact, in both these techniques large crystals can prove problematic. This is because large crystals can clog up the nozzle of certain SFX sample delivery systems6,7, while in MicroED the large electron scattering cross-section implies that absorption extinguishes diffraction when the sample is too thick8.
Author Manuscript Author Manuscript
Here we show that sonication, vigorous pipetting, or vortexing can be used to break large imperfect crystals into small, single-crystal fragments that are suitable for data collection and atomic structure determination (Figure 2). Delicate samples may benefit from gentler fragmentation by vortexing, while harsher methods such as pipetting and ultimately sonication are required to break more robust crystals. In many cases, the untreated crystals were mosaic, yielded diffraction patterns with multiple lattices, or were otherwise not suitable for standard crystallographic experiments (Figure 2, Supplementary figures 1–9). In the case of the amyloid-forming peptide of tau (VQIVYK), what appeared to be large crystals were in fact crystal bundles that produced low resolution powder-like diffraction. These problems are traditionally overcome by modifying the crystallization conditions to optimize crystal growth and quality a process that can be tedious and labor intensive, particularly when crystal pathologies do not become apparent until data processing. Breaking of large crystals by physical means produced fragments that appeared crystallographically homogeneous and yielded diffraction data at atomic resolution, void of the above artifacts. This was true even without further optimization of the growth conditions for crystals formed by various macromolecules of molecular weights between 0.7 kDa and 34.6 kDa, and solvent contents ranging from 30% to 60% with a range of unit cell dimensions and space groups (Table 1). MicroED has already allowed rapid structure solution from several peptide fragments that resisted solution using microfocus synchrotron sources in spite of many months of crystal optimization (Supplementary figures 2–4), and the approach could be used for samples where large crystals exist but standard crystallographic methods fail, owing to crystal imperfections. Fragmentation was tested on eight proteins: lysozyme, TGF-βm:TβRII, xylanase, thaumatin, trypsin, proteinase K, thermolysin, and a segment of the protein tau (Figure 2, left column). These proteins readily form crystals that are large—too big for MicroED and SFX experiments using liquid injectors—but without crystal growth optimization sometimes exhibit pathologies. These large crystals were broken apart by mechanical means prior to preparation on cryoEM grids. Micro- or nanometer-sized crystal fragments appeared evenly
Nat Methods. Author manuscript; available in PMC 2017 August 13.
de la Cruz et al.
Page 3
Author Manuscript
distributed on the grid, and even when grids were densely populated single-crystal data sets could be collected by using the selective area aperture. MicroED is inherently well suited to study crystal fragments. Electrons interact strongly with matter so that large crystals are not required to detect high-resolution Bragg reflections8. MicroED has been used to determine the structures of the enzymes lysozyme5,9, proteinase K10, catalase11,12, and Ca2+-ATPase12. It was also recently used to solve the 1.4 Å resolution structure of the toxic NAC-core of α-synuclein, where diffraction data were collected from crystals smaller than the wavelength of visible light13. We previously demonstrated that crystals thinner than ~400 nm are suitable for MicroED and routinely yield atomic resolution information. The highest resolution structures reported so far by using MicroED are from four prion protein fragments determined at 1 Å resolution solved by direct phasing methods14.
Author Manuscript Author Manuscript
For each sample, data were collected from 2–10 crystal fragments, and data sets merged for completeness and multiplicity (Table 1). Although we have previously published two structures where a single nanocrystal was sufficient for structure determination9,11 multicrystal merging is generally preferred. This is partly because the grid on which the crystals are mounted limits the accessible rotation range during data collection, and partly because small, weakly scattering samples require a more intense beam to yield statistically accurate measurements of the Bragg reflections, and may be irreversibly damaged before a complete data set can be obtained. Recording narrow wedges with fewer exposures for each crystal involves a tradeoff between limiting the damage to the sample and maintaining a sufficient dose rate for the signal from high-resolution reflections to be accurately integrated. Because the resolutions obtained by MicroED are comparable or better to what was obtained by Xray diffraction before sonication, it appears that fragmentation only broke apart the large crystals into small crystal domains but did not otherwise damage the lattice order.
Author Manuscript
The macromolecular structures in this study were phased using molecular replacement (MR) or by direct methods and refined using electron scattering factors15. In all cases, the refined model fits the calculated density well (Figure 2, middle column) with an overall real-space map correlation coefficient ranging from 0.72 to 0.91 (RSCC; Table 1). The resulting simulated-annealing (SA) composite-omit maps (Figure 2, right column) match their respective models very well, indicating that the data are indeed high quality and unbiased. Further, the SA omit maps for lysozyme reveal depressions or holes in the aromatic rings of amino acid side chains, while for proteinase K toroidal densities were observed even for proline residues at the 1.6 Å resolution cutoff. A well-coordinated calcium ion is clearly visible in the omit map for trypsin, as is one of the iodides in the xylanase structure. Individual atoms are visible in the density for the tau peptide, and several residues show positive 2mFo-DFc peaks for hydrogen atoms; for one residue, modeled hydrogens allowed us to unambiguously place the correct sidechain rotamer. All structures were determined between 1.1–2.9 Å resolution. We note that the R-factors from model refinement are generally higher in MicroED than typical values obtained in Xray crystallography at similar resolutions. Others and we have already made such observations in previously published studies5,9,12. We believe that the main reason is lack of
Nat Methods. Author manuscript; available in PMC 2017 August 13.
de la Cruz et al.
Page 4
Author Manuscript
adequate electron scattering factors in the crystallographic refinement software. X-rays are scattered by the electron cloud of an atom while electrons are scattered by the atomic Coulomb potential, which arises from the nucleus as well as its surrounding electrons. Current electron scattering factor tables do not adequately account for this difference and this may contribute to the residual between observed and calculated structure factor amplitudes leading to higher than normal crystallographic and free R-factors. We note that the gap between Rwork and Rfree is small indicating no concerns of overfitting (Supplementary figure 10).
Discussion
Author Manuscript
Breaking large crystals into well-formed crystal fragments has a long history in crystallography; indeed X-ray diffraction was discovered from broken up pieces of a copper sulfate crystal16. Sonication17 and vortexing18 have previously been used to prepare crystals of suitable size for XFEL measurements. By breaking up one imperfect large crystal into thousands or even millions of smaller crystallites and recombining a well-diffracting subset during data processing, we have used MicroED to determine the structure of eight different proteins to high resolution. Fragmentation does not increase the effort required for sample screening, and future work on automation is expected to reduce the time for data collection irrespective of how the sample was prepared. Importantly, as demonstrated by the breadth of size and packing of macromolecules whose crystals we investigated, this is a broadly applicable approach where large crystals are available. Fragmentation widens the scope of diffraction methods such as MicroED and SFX to include samples that do not exclusively form tiny crystals but instead form large, imperfect crystals.
Author Manuscript
Online methods Large crystals (>500 µm along the longest edge) were grown via hanging-drop vapor diffusion at room temperature using previously established protocols. All enzymes were purchased from Sigma-Aldrich (St. Louis, MO) unless otherwise noted.
Author Manuscript
•
Tau peptide (VQIVYK) was dissolved in distilled water and crystals prepared by mixing tau peptide with arachidonic acid and meclocycline sulfosalicylate. Crystals formed with a 1:2 drop peptide to precipitant ratio from 65–70% ethylene glycol in Tris pH 8.5.
•
Lysozyme (G. gallus) crystals were prepared by equilibrating equal volumes of 50 mg/ml lysozyme in water and 1.7 M sodium chloride, 50 mM sodium acetate pH 4.7.
•
TGF-βm:TβRII was expressed and purified as described before20,21. Crystals were grown from 0.5 µl of protein (20 mg/ml), 0.25 µl mother liquor, and 0.2 µl seed stock in 100 mM HEPES/NaOH pH 7.5 and 45% MPD.
•
Xylanase from T. reesei (Hampton Research, Aliso Viejo, CA) was dialyzed against 10 mM bicine pH 9.0, 1 mM magnesium sulfate, 1 mM DTT and combined with a precipitant solution containing 0.3 M sodium iodide, 1.2–1.3 M ammonium sulfate, and 100 mM bicine pH 9.022.
Nat Methods. Author manuscript; available in PMC 2017 August 13.
de la Cruz et al.
Page 5
Author Manuscript Author Manuscript
•
Thaumatin (T. daniellii) crystals were grown from 2 µL of protein (25 mg/ml in water) and Hampton Research Index Reagent 26 (1.1 M ammonium tartate pH 7.0).
•
Trypsin (B. taurus) was dissolved (60 mg/ml) in 10 mg/ml benzamidine, 3 mM calcium chloride and equilibrated against 4% (w/v) PEG4000, 0.2 M lithium sulfate, 0.1 M MES pH 6.5 and 15% ethylene glycol.
•
Proteinase K (E. album) crystals were grown by combining 2 µL of protein solution (50 mg/ml) with 2 µL of precipitant solution (1.0–1.3 M ammonium sulfate, 0.1 M Tris pH 8.0)10.
•
Thermolysin from B. thermoproteolyticus (Hampton Research, Aliso Viejo, CA) crystals were prepared by equilibrating a 160 mg/ml solution of thermolysin (45% dimethyl sulfoxide, 50 mM Tris pH 7.5 and 2.5 M cesium chloride) over 0.5 ml of water23.
Drops containing the crystals were placed in separate microfuge tubes and suspended in crystal mother liquor. A sonicating water bath with electronic control (Elmasonic P30H, Singen, Germany) was set at its lowest power (30% at 37 kHz) for gentle agitation of the crystals in the tube. With the tube sealed, its tip was briefly submerged in the activated water bath for 0.5 s. Alternatively; crystals were fragmented by vigorously pipetting a crystal suspension in mother liquor (trypsin and thaumatin) or vortexed with 0.5 mm disruption glass beads in a 1.5 ml reaction tube for 2 s (TGF-βm:TβRII). A detailed protocol for crystal fragmentation was published online24.
Author Manuscript Author Manuscript
The solution containing fragmented crystals was then applied to transmission electron microscope (TEM) grids with carbon film support, and plunge-frozen in liquid ethane. Frozen grids were mounted in a Gatan Model 626 cryo-specimen holder and examined using an FEI Tecnai F20 field-emission TEM operated at an accelerating voltage of 200 kV, which corresponds to a de Broglie wavelength of 0.025 Å, and screened for crystals in overfocused diffraction mode. Where a single still shot revealed strong diffraction, data were collected as continuous rotation tilt series9. Individual frames were recorded on a TVIPS TemCam-F416 as 4 s exposures while the stage was rotating at 0.09°/s, except for tau peptide, which was rotated at 0.29°/s during 2 s exposures. For the macromolecular samples, the absolute tilt angle was generally
